The Chd1 chromatin remodeler forms long-lived complexes with nucleosomes in the presence of ADP·BeF3- and transition state analogs.

Chromatin remodelers use helicase-like ATPase domains to reorganize histone-DNA contacts within the nucleosome. Like other remodelers, the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler repositions nucleosomes by altering DNA topology at its internal binding site on the nucleosome, coupling different degrees of DNA twist and DNA movement to distinct nucleotide-bound states of the ATPase motor. In this work, we used a competition assay to study how variations in the bound nucleotide, Chd1, and the nucleosome substrate affect stability of Chd1-nucleosome complexes. We found that Chd1-nucleosome complexes formed in nucleotide-free or ADP conditions were relatively unstable and dissociated within 30 s, whereas those with the nonhydrolyzable ATP analog AMP-PNP had a mean lifetime of 4.8 ± 0.7 min. Chd1-nucleosome complexes were remarkably stable with ADP·BeF3- and the transition state analogs ADP·AlFX and ADP·MgFX, being resistant to competitor nucleosome over a 24-h period. For the tight ADP·BeF3--stabilized complex, Mg2+ was a critical component that did not freely exchange, and formation of these long-lived complexes had a slow, concentration-dependent step. The ADP·BeF3--stabilized complex did not require the Chd1 DNA-binding domain nor the histone H4 tail and appeared relatively insensitive to sequence differences on either side of the Widom 601 sequence. Interestingly, the complex remained stable in ADP·BeF3- even when nucleosomes contained single-stranded gaps that disrupted most DNA contacts with the guide strand. This finding suggests that binding via the tracking strand alone is sufficient for stabilizing the complex in a hydrolysis-competent state.

Reb1, Cbf1, and Pho4 Bias Histone Sliding and Deposition Away from Their Binding Sites.

In transcriptionally active genes, nucleosome positions in promoters are regulated by nucleosome-displacing factors (NDFs) and chromatin-remodeling enzymes. Depletion of NDFs or the RSC chromatin remodeler shrinks or abolishes the nucleosome-depleted regions (NDRs) in promoters, which can suppress gene activation and result in cryptic transcription. Despite their vital cellular functions, how the action of chromatin remodelers may be directly affected by site-specific binding factors like NDFs is poorly understood. Here, we demonstrate that two NDFs, Reb1 and Cbf1, can direct both Chd1 and RSC chromatin-remodeling enzymes in vitro, stimulating repositioning of the histone core away from their binding sites. Interestingly, although the Pho4 transcription factor had a much weaker effect on nucleosome positioning, both NDFs and Pho4 were able to similarly redirect positioning of hexasomes. In chaperone-mediated nucleosome assembly assays, Reb1 but not Pho4 showed an ability to block deposition of the histone H3/H4 tetramer, but Reb1 did not block addition of the H2A/H2B dimer to hexasomes. Our in vitro results show that NDFs bias the action of remodelers to increase the length of the free DNA in the vicinity of their binding sites. These results suggest that NDFs could directly affect NDR architecture through chromatin remodelers.

Chd1 co-localizes with early transcription elongation factors independently of H3K36 methylation and releases stalled RNA polymerase II at introns.

BACKGROUND: Chromatin consists of ordered nucleosomal arrays that are controlled by highly conserved adenosine triphosphate (ATP)-dependent chromatin remodeling complexes. One such remodeler, chromodomain helicase DNA binding protein 1 (Chd1), is believed to play an integral role in nucleosomal organization, as the loss of Chd1 is known to disrupt chromatin. However, the specificity and basis for the functional and physical localization of Chd1 on chromatin remains largely unknown. RESULTS: Using genome-wide approaches, we found that the loss of Chd1 significantly disrupted nucleosome arrays within the gene bodies of highly transcribed genes. We also found that Chd1 is physically recruited to gene bodies, and that its occupancy specifically corresponds to that of the early elongating form of RNA polymerase, RNAPII Ser 5-P. Conversely, RNAPII Ser 5-P occupancy was affected by the loss of Chd1, suggesting that Chd1 is associated with early transcription elongation. Surprisingly, the occupancy of RNAPII Ser 5-P was affected by the loss of Chd1 specifically at intron-containing genes. Nucleosome turnover was also affected at these sites in the absence of Chd1. We also found that deletion of the histone methyltransferase for H3K36 (SET2) did not affect either Chd1 occupancy or nucleosome organization genome-wide. CONCLUSIONS: Chd1 is specifically recruited onto the gene bodies of highly transcribed genes in an elongation-dependent but H3K36me3-independent manner. Chd1 co-localizes with the early elongating form of RNA polymerase, and affects the occupancy of RNAPII only at genes containing introns, suggesting a role in relieving splicing-related pausing of RNAPII.